Protein-solvent interfaces in human Y145Stop prion protein amyloid fibrils probed by paramagnetic solid-state NMR spectroscopy.

The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13C,15N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy. To further probe the interactions of the amyloid core residues with solvent molecules we perform complementary measurements of amide hydrogen/deuterium exchange detected by solid-state NMR and solution NMR methods. The solvent accessibility data are evaluated in the context of the structural model for human PrP23-144 amyloid.

Species-dependent structural polymorphism of Y145Stop prion protein amyloid revealed by solid-state NMR spectroscopy.

One of the most puzzling aspects of the prion diseases is the intricate relationship between prion strains and interspecies transmissibility barriers. Previously we have shown that certain fundamental aspects of mammalian prion propagation, including the strain phenomenon and species barriers, can be reproduced in vitro in seeded fibrillization of the Y145Stop prion protein variant. Here, we use solid-state nuclear magnetic resonance spectroscopy to gain atomic level insight into the structural differences between Y145Stop prion protein amyloids from three species: human, mouse, and Syrian hamster. Remarkably, we find that these structural differences are largely controlled by only two amino acids at positions 112 and 139, and that the same residues appear to be key to the emergence of structurally distinct amyloid strains within the same protein sequence. The role of these residues as conformational switches can be rationalized based on a model for human Y145Stop prion protein amyloid, providing a foundation for understanding cross-seeding specificity.Prion diseases can be transmitted across species. Here the authors use solid-state NMR to study prion protein (PrP) amyloids from human, mouse and Syrian hamster and show that their structural differences are mainly governed by two residues, which helps to understand interspecies PrP propagation on a molecular level.

Mechanism of Nucleated Conformational Conversion of Aß42.

Soluble oligomers and protofibrils of the Aß42 peptide are neurotoxic intermediates in the conversion of monomeric Aß42 into the amyloid fibrils associated with Alzheimer's disease. Nuclear magnetic resonance and Fourier transform infrared spectroscopy, along with single-touch atomic force microscopy, are used to establish the structural transitions involved in fibril formation. We show that under conditions favorable for the nucleated conformation conversion, the Aß42 peptide aggregates into largely unstructured low-molecular weight (MW) oligomers that are able to stack to form high-MW oligomers and to laterally associate to form protofibrils. ß-Sheet secondary structure develops during the irreversible lateral association of the oligomers. The first step in this conversion is the formation of an antiparallel ß-hairpin stabilized by intramonomer hydrogen bonding. The antiparallel ß-hairpins then associate into a cross ß-sheet structure with parallel and in-register ß-strands having intermonomer hydrogen bonding.

Capping of aß42 oligomers by small molecule inhibitors.

Aß42 peptides associate into soluble oligomers and protofibrils in the process of forming the amyloid fibrils associated with Alzheimer's disease. The oligomers have been reported to be more toxic to neurons than fibrils, and have been targeted by a wide range of small molecule and peptide inhibitors. With single touch atomic force microscopy (AFM), we show that monomeric Aß42 forms two distinct types of oligomers, low molecular weight (MW) oligomers with heights of 1-2 nm and high MW oligomers with heights of 3-5 nm. In both cases, the oligomers are disc-shaped with diameters of ~10-15 nm. The similar diameters suggest that the low MW species stack to form the high MW oligomers. The ability of Aß42 inhibitors to interact with these oligomers is probed using atomic force microscopy and NMR spectroscopy. We show that curcumin and resveratrol bind to the N-terminus (residues 5-20) of Aß42 monomers and cap the height of the oligomers that are formed at 1-2 nm. A second class of inhibitors, which includes sulindac sulfide and indomethacin, exhibit very weak interactions across the Aß42 sequence and do not block the formation of the high MW oligomers. The correlation between N-terminal interactions and capping of the height of the Aß oligomers provides insights into the mechanism of inhibition and the pathway of Aß aggregation.

Fine mapping of the amyloid ß-protein binding site on myelin basic protein.

The assembly and deposition of amyloid ß-protein (Aß) in brain is a key pathological feature of Alzheimer's disease and related disorders. Factors have been identified that can either promote or inhibit Aß assembly in brain. We previously reported that myelin basic protein (MBP) is a potent inhibitor of Aß fibrillar assembly [Hoos, M. D., et al. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., et al. (2009) Biochemistry 48, 4720-4727]. Moreover, the region on MBP responsible for this activity was localized to the 64 N-terminal amino acids (MBP1-64) [Liao, M. C., et al. (2010) J. Biol. Chem. 285, 35590-35598]. In the study presented here, we sought to further define the site on MBP1-64 involved in this activity. Deletion mapping studies showed that the C-terminal region (residues 54-64) is required for the ability of MBP1-64 to bind Aß and inhibit fibril assembly. Alanine scanning mutagenesis revealed that amino acids K54, R55, G56, and K59 within MBP1-64 are important for both Aß binding and inhibition of fibril assembly as assessed by solid phase binding, thioflavin T binding and fluorescence, and transmission electron microscopy studies. Strong spectral shifts are observed by solution nuclear magnetic resonance spectroscopy of specific N-terminal residues (E3, R5, D7, E11, and Q15) of Aß42 upon the interaction with MBP1-64. Although the C-terminal region of MBP1-64 is required for interactions with Aß, a synthetic MBP50-64 peptide was itself devoid of activity. These studies identify key residues in MBP and Aß involved in their interactions and provide structural insight into how MBP regulates Aß fibrillar assembly.

Conformational differences between two amyloid ß oligomers of similar size and dissimilar toxicity.

Several protein conformational disorders (Parkinson and prion diseases) are linked to aberrant folding of proteins into prefibrillar oligomers and amyloid fibrils. Although prefibrillar oligomers are more toxic than their fibrillar counterparts, it is difficult to decouple the origin of their dissimilar toxicity because oligomers and fibrils differ both in terms of structure and size. Here we report the characterization of two oligomers of the 42-residue amyloid ß (Aß42) peptide associated with Alzheimer disease that possess similar size and dissimilar toxicity. We find that Aß42 spontaneously forms prefibrillar oligomers at Aß concentrations below 30 µm in the absence of agitation, whereas higher Aß concentrations lead to rapid formation of fibrils. Interestingly, Aß prefibrillar oligomers do not convert into fibrils under quiescent assembly conditions but instead convert into a second type of oligomer with size and morphology similar to those of Aß prefibrillar oligomers. Strikingly, this alternative Aß oligomer is non-toxic to mammalian cells relative to Aß monomer. We find that two hydrophobic peptide segments within Aß (residues 16-22 and 30-42) are more solvent-exposed in the more toxic Aß oligomer. The less toxic oligomer is devoid of ß-sheet structure, insoluble, and non-immunoreactive with oligomer- and fibril-specific antibodies. Moreover, the less toxic oligomer is incapable of disrupting lipid bilayers, in contrast to its more toxic oligomeric counterpart. Our results suggest that the ability of non-fibrillar Aß oligomers to interact with and disrupt cellular membranes is linked to the degree of solvent exposure of their central and C-terminal hydrophobic peptide segments.

Mechanistic studies of peptide self-assembly: transient α-helices to stable ß-sheets.

The pathologic self-assembly of proteins is associated with typically late-onset disorders such as Alzheimer's disease, Parkinson's disease, and type 2 diabetes. Important mechanistic details of the self-assembly are unknown, but there is increasing evidence supporting the role of transient α-helices in the early events. Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide that self-assembles into aggregates that are toxic to the insulin-producing ß cells. To elucidate early events in the self-assembly of IAPP, we used limited proteolysis to identify an exposed and flexible region in IAPP monomer. This region includes position 20 where a serine-to-glycine substitution (S20G) is associated with enhanced formation of amyloid fibrils and early onset type 2 diabetes. To perform detailed biophysical studies of the exposed and flexible region, we synthesized three peptides including IAPP(11-25)WT (wild type), IAPP(11-25)S20G, and IAPP(11-25)S20P. Solution-state NMR shows that all three peptides transiently populate the α-helical conformational space, but the S20P peptide, which does not self-assemble, transiently samples a broken helix. Under similar sample conditions, the WT and S20G peptides populate the α-helical intermediate state and ß-sheet end state, respectively, of fibril formation. Our results suggest a mechanism for self-assembly that includes the stabilization of transient α-helices through the formation of NMR-invisible helical intermediates followed by an α-helix to ß-sheet conformational rearrangement. Furthermore, our results suggest that reducing intermolecular helix-helix contacts as in the S20P peptide is an attractive strategy for the design of blockers of peptide self-assembly.

N-terminal domain of myelin basic protein inhibits amyloid beta-protein fibril assembly.

Accumulation of amyloid ß-protein (Aß) into brain parenchymal plaques and the cerebral vasculature is a pathological feature of Alzheimer disease and related disorders. Aß peptides readily form ß-sheet-containing oligomers and fibrils. Previously, we reported a strong interaction between myelin basic protein (MBP) and Aß peptides that resulted in potent inhibition of fibril assembly (Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., Ahmed, M., Smith, S. O., and Van Nostrand, W. E. (2009) Biochemistry 48, 4720-4727). MBP is recognized as a highly post-translationally modified protein. In the present study, we demonstrate that human MBP purified from either brain or a bacterial recombinant expression system comparably bound to Aß and inhibited Aß fibril assembly indicating that post-translational modifications are not required for this activity. We also show that purified mouse brain MBP and recombinantly expressed mouse MBP similarly inhibited Aß fibril formation. Through a combination of biochemical and ultrastructural techniques, we demonstrate that the binding site for Aß is located in the N-terminal 64 amino acids of MBP and that a stable peptide (MBP1) comprising these residues was sufficient to inhibit Aß fibrillogenesis. Under conditions comparable with those used for Aß, the fibrillar assembly of amylin, another amyloidogenic peptide, was not inhibited by MBP1, although MBP1 still bound to it. This observation suggests that the potent inhibitory effect of MBP on fibril formation is not general to amyloidogenic peptides. Finally, MBP1 could prevent the cytotoxic effects of Aß in primary cortical neurons. Our findings suggest that inhibition of Aß fibril assembly by MBP, mediated through its N-terminal domain, could play a role in influencing amyloid formation in Alzheimer disease brain and corresponding mouse models.

Structural conversion of neurotoxic amyloid-beta(1-42) oligomers to fibrils.

The amyloid-beta(1-42) (Abeta42) peptide rapidly aggregates to form oligomers, protofibils and fibrils en route to the deposition of amyloid plaques associated with Alzheimer's disease. We show that low-temperature and low-salt conditions can stabilize disc-shaped oligomers (pentamers) that are substantially more toxic to mouse cortical neurons than protofibrils and fibrils. We find that these neurotoxic oligomers do not have the beta-sheet structure characteristic of fibrils. Rather, the oligomers are composed of loosely aggregated strands whose C termini are protected from solvent exchange and which have a turn conformation, placing Phe19 in contact with Leu34. On the basis of NMR spectroscopy, we show that the structural conversion of Abeta42 oligomers to fibrils involves the association of these loosely aggregated strands into beta-sheets whose individual beta-strands polymerize in a parallel, in-register orientation and are staggered at an intermonomer contact between Gln15 and Gly37.

High-resolution 1H MAS RFDR NMR of biological membranes.

The combination of magic angle spinning (MAS) with the high-resolution (1)H NOESY NMR experiment is an established method for measuring through-space (1)H...(1)H dipolar couplings in biological membranes. The segmental motion of the lipid acyl chains along with the overall rotational diffusion of the lipids provides sufficient motion to average the (1)H dipolar interaction to within the range where MAS can be effective. One drawback of the approach is the relatively long NOESY mixing times needed for relaxation processes to generate significant crosspeak intensity. In order to drive magnetization transfer more rapidly, we use solid-state radiofrequency driven dipolar recoupling (RFDR) pulses during the mixing time. We compare the (1)H MAS NOESY experiment with a (1)H MAS RFDR experiment on dimyristoylphosphocholine, a bilayer-forming lipid and show that the (1)H MAS RFDR experiment provides considerably faster magnetization exchange than the standard (1)H MAS NOESY experiment. We apply the method to model compounds containing basic and aromatic amino acids bound to membrane bilayers to illustrate the ability to locate the position of aromatic groups that have penetrated to below the level of the lipid headgroups.